1. Field of the Invention
The present invention relates to the fields of molecular biology and oncology. Specifically, the present invention relates to, inter alia, methods for using antisense oligonucleotides to inhibit microRNA-27a suppression of the Sp repressor ZBTB10 in cancer cells.
2. Description of the Related Art
Specificity protein 1 (Sp1) was the first transcription factor identified (1,2) and is a member of the Sp/Kruppel-like factor (KLF) family of transcription factors (3-6). These proteins are characterized by their C-terminal domains which contain three C2H2-type zinc fingers that recognize GC/GT boxes in promoters of target genes. The N-terminal domains of Sp/KLF proteins are highly variable in both structure and function and many KLF proteins are truncated in this region. Some Sp/KLF members are critical for embryonic development and knockout of Sp1, Sp3 and Sp4 genes in mice results in embryo lethality or multiple developmental deficits (7-10). Sp and KLF proteins cooperatively interact with one another and other transcription factors on GC-rich promoters to activate or inhibit diverse classes of mammalian and viral genes that play a critical role in regulating cellular homeostasis (11).
The tissue- and age-dependent expression of Sp proteins in humans and laboratory animal models has not been extensively investigated, however, several studies report that Sp1 protein is overexpressed in tumor vs. non-tumor tissues (12-19). For example, in gastric cancer Sp1 expression was observed in tumor cells; whereas in stromal and normal glandular cells, Sp1 expression was either weak or non-detectable (12). Moreover, survival of gastric cancer patients increased with decreasing Sp1 protein expression. Malignant transformation of human fibroblasts resulted in an 8- to 18-fold increase in Sp1 expression and the transformed cells formed tumors in athymic nude mouse xenografts, whereas Sp1 knockdown gave cells that were non-tumorigenic in the same mouse xenograft model (19).
Although Sp1 is widely expressed in tumors, there is increasing evidence that Sp3 and Sp4 also are expressed in cancer cells and contribute to Sp dependent procarcinogenic responses (20-25). Using RNA interference, it was shown that Sp1 knockdown using a small inhibitory RNA (siRNA) for Sp1 (iSp1) inhibited G0/G1 to S phase progression in MCF-7 breast cancer cells (25). SiRNAs for Sp3 (iSp3) and Sp4 (iSp4) were used along with iSp1 to show that in pancreatic cancer cells, Sp1, Sp3 and Sp4 proteins regulated expression of vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR1 or Flt) and VEGFR2 (KDR) (20-22). Moreover, Sp3 acted as a repressor of p27 in pancreatic cancer cells (21), indicating that overexpression of Sp proteins in cancers contribute to their proliferative and angiogenic phenotype. The underlying factors associated with high expression of Sp proteins, such as Sp1, Sp3 and Sp4 in tumors are not well understood.
The underlying factors associated with high expression of Sp proteins such as Sp1, Sp3 and Sp4 in tumors are not well understood. It is contemplated that microRNAs (miRNAs) may play a role in mediating overexpression of these transcription factors in tumors and cancer cells. mRNAs are small noncoding RNAs that regulate expression of genes by specifically interacting with 3′-untranslated regions of target gene mRNAs to repress translation or enhance mRNA cleavage (26, 27). It was observed that miRNA-27a (miR-27a) may have affected ZBTB10/RINZF expression; however, the extent of this interaction was not quantitated (28). This novel zinc finger protein (ZBTB10) inhibits activation of the GC-rich gastrin gene promoter (29).
There is a recognized need in the art for improved cancer therapies. Specifically, the prior art is deficient in methods utilizing antisense or related technologies to inhibit miR-27a suppression genes such as ZBTB10 which would repress overexpression of Sp proteins and other Sp-dependent genes/proteins in tumors or cancer cells. The present invention fulfills this long standing need in the art.